Usefulness of cardiac hormones for evaluating valvular disease in cynomolgus monkeys (Macaca fascicularis).

Nonhuman primates are commonly used as experimental animals due to their biological resemblance to humans. In patients with cardiac disease, the levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) tend to increase in response to cardiac damage, and they are thus used as indicators for the diagnosis of human heart failure. However, no reference values for ANP and BNP have been reported for heart disease in nonhuman primates. In this study, we recorded the age, sex, and body weight of 202 cynomolgus monkeys, and performed evaluations to assess the ANP and BNP levels, electrocardiography and echocardiography, and accordingly divided the monkeys into two groups: healthy monkeys and those with spontaneous cardiac disease. Statistical analysis was performed to determine the relationship of ANP and BNP with the factors of age, sex, and body weight. No significant relationship was found between the levels of ANP and BNP and the factors of age, sex, and body weight. However, both the ANP and BNP levels were significantly different between the healthy monkeys and monkeys with valvular disease. Similar to humans, the ANP and BNP levels tended to increase with the progression of cardiac disease in monkeys. Based on these results, we concluded that ANP and BNP are indicators of cardiac disease in nonhuman primates, and that this nonhuman primate cardiac disease model is applicable for cardiology research in humans.

Echocardiographic evaluation of cardiac function in cynomolgus monkeys over a wide age range.

Various cardiovascular diseases can be detected and diagnosed using echocardiography. The demand for cardiovascular system research using nonhuman primates is increasing, but echocardiographic references for nonhuman primates are limited. This report describes the first comparison of echocardiographic reference values in 247 normal cynomolgus monkeys (135 females, 112 males) over a wide age range. Echocardiography, electrocardiography, blood pressure and chest X-ray images were acquired under immobilization with intramuscular ketamine hydrochloride, then cardiac structure, function, and flow velocity were assessed. Cardiac hormone levels were also tested. We found that cardiac structures positively correlated with weight, that the size of these structures stabilized after reaching maturity and that cardiac output increased according to heart size. In contrast, fractional shortening of the left ventricle, ejection fraction and flow velocity showed no significant correlations with weight or age, and age and E wave correlated negatively. These findings appear sufficiently similar to those in humans to suggest that cynomolgus monkeys can serve as a suitable model of human cardiac disease. Our data should also prove useful for surveying cardiac dysfunction in monkeys.

Establishment of a new formula for QT interval correction using a large colony of cynomolgus monkeys.

The demand for monkeys for medical research is increasing, because their ionic mechanism of repolarization is similar to that of humans. The QT interval is the distance between the Q wave and T wave, but this interval is affected by heart rate. Therefore, QT correction methods are commonly used in clinical settings. However, an accurate correction formula for the QT interval in cynomolgus monkeys has not been reported. We assessed snapshot electrocardiograms (ECGs) of 353 ketamine-immobilized monkeys, including aged animals, and contrived a new formula for the corrected QT interval (QTc) as a marker of QT interval prolongation in cynomolgus monkeys. Values for QTc were calculated using the formula [QTc] = [QT] / [RR]n, along with several other formulas commonly used to calculate QTc. We found that the optimal exponent of the QT interval corrected for heart rate, n, was 0.576. The mean value of QTc in healthy monkeys determined using the new formula was 373 ± 31 mm, and there were no significant differences between the sexes. Other ECG parameters were not significantly different between the sexes and there were no age-related effects on QTc. Prolongation of QTc to over 405 ms, as calculated by the new formula, was observed in 50 monkeys with underlying diseases. Additionally, all monkeys with QTc above 440 ms by the new formula had some underlying disease. The results resemble those in humans, suggesting that the new QTc formula could be useful for diagnosis of QT interval prolongation in cynomolgus monkeys.

Utility of arterial blood gas, CBC, biochemistry and cardiac hormones as evaluation parameters of cardiovascular disease in nonhuman primates.

Cardiovascular disease (CVD) has a tremendous impact on the quality of life of humans. While experimental animals are valuable to medical research as models of human diseases, cardiac systems differ widely across various animal species. Thus, we examined a CVD model in cynomolgus monkeys. Laboratory primates are precious resources, making it imperative that symptoms of diseases and disorders are detected as early as possible. Thus, in this study we comprehensively examined important indicators of CVD in cynomolgus monkeys, including arterial blood gas, complete blood count (CBC), biochemistry and cardiac hormones. The control group included 20 healthy macaques showing non-abnormal findings in screening tests, whereas the CVD group included 20 macaques with valvular disease and cardiomyopathy. An increase of red blood cell distribution width was observed in the CBC, indicating chronic inflammation related to CVD. An increase of HCO3 was attributed to the correction of acidosis. Furthermore, development of the CVD model was supported by significant increases in natriuretic peptides. It is suggested that these results indicated a correlation between human CVD and the model in monkeys. Moreover, blood tests including arterial blood gas are non-invasive and can be performed more easily than other technical tests. CVD affected animals easily change their condition by anesthesia and surgical invasion. Pay attention to arterial blood gas and proper respond to their condition are important for research. This data may facilitate human research and aid in the management and veterinary care of nonhuman primates.

Superparamagnetic Iron Oxide-enhanced Magnetic Resonance Imaging of Spontaneous Hepatic Neoplasia in a Cynomolgus Macaque (Macaca fascicularis).

Although the number of reports describing tumors in aged NHP has increased, spontaneous neoplasias in NHP are extremely rare, with the notable exception of prosimians, in which spontaneous hepatic neoplasms arise. In addition to radiography and ultrasonography, superparamagnetic iron oxide (SPIO)-enhanced MRI tends to be applied in human practice to non-invasively locate, identify, and size liver tumors and to define the border between neoplastic and normal tissues. Here we report a 13-y-old female cynomolgus monkey with anorexia and serologically normal liver enzymes. After fluid therapy, the condition remained in remission for several months. Later, however, a palpable mass was assessed by using ultrasonography, radiology, and SPIO-MRI; T2-weighted images revealed a clear border between a hepatocellular carcinoma and normal liver tissue. Findings at necropsy supported the imaging data. Serologic assessment after euthanasia revealed a positive reaction to an abnormal form of prothrombin (PIVKA-II). We recommend SPIO-MRI as a practical and useful for diagnosing hepatocellular neoplasias in NHP. This study is the first to demonstrate the applicability of SPIO-MRI for the identification of hepatocellular carcinoma in NHP.

Establishment of reference values for complete blood count and blood gases in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys are closely related to humans phylogenetically, and this has resulted in their widespread use as a preclinical model. Hematological data with regard to these monkeys are thus important. Although reference values for blood components and sex hormones have been established for cynomolgus monkeys, those for arterial blood gases have not. The arterial blood gases quickly reflect respiratory and circulatory dynamics, and are thus useful for animal management and safe general anesthesia and surgical operations. Furthermore, since O2 is transported by RBC, CBC and blood gases are closely related. The present study aimed to establish reference values for arterial blood gases and CBC in cynomolgus monkeys over a wide age range. Blood gases and CBC of arterial blood, collected from 41 female and 21 male anesthetized monkeys, were measured. Age correlated with RBC, HGB and HCT in the CBC. Values differed significantly between males and females in pCO2, CO2 concentration, MCV and MCH. The pH of blood was equivalent to that of humans and pCO2 was more stable, whereas MCV and MCH were lower than those in humans. Erythrocytes were smaller and less pigmented than in other Macaca species. Several relationships between gender and age, and blood gases and CBC were identified in cynomolgus monkeys. In conclusion, these reference values will be useful as markers for veterinary applications and in the care and maintenance of these animals.

Tracking cells implanted into cynomolgus monkeys (Macaca fascicularis) using MRI.

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger's disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy.

Analysis of the breast milk of giant pandas (Ailuropoda melanoleuca) and the preparation of substitutes.

The first milk substitute for giant panda cubs was developed in 1988 based on limited data about giant panda breast milk and that of certain types of bear. Mixtures of other formulas have also been fed to cubs at some facilities. However, they are not of sufficient nutritional quality for promoting growth in panda cubs. Here, we report analysis of giant panda breast milk and propose new milk substitutes for cubs, which were developed based on the results of our analysis. The Chengdu Research Base of Giant Panda Breeding obtained breast milk samples from three giant pandas. Up to 30 ml of breast milk were collected from each mother by hand. Then, the milk samples were frozen and sent to Nihon University. The levels of protein, fat, carbohydrates, ash, moisture, vitamins, minerals, total amino acids, fatty acids, lactose and other carbohydrates in the milk were analyzed. The breast milk samples exhibited the following nutritional values: protein: 6.6-8.5%, fat: 6.9-16.4%, carbohydrates: 2.5-9.1%, ash: 0.9-1.0% and moisture: 67-83%. We designed two kinds of milk substitutes based on the data obtained and the nutritional requirements of dogs, cats and rodents. The nutritional composition of the milk substitutes for the first and second stages was as follows: protein: 38 and 26%, fat: 40 and 40%, carbohydrates: 13 and 25%, ash: 6 and 6% and moisture: 3 and 3%, respectively. In addition, the substitutes contained vitamins, minerals, taurine, docosahexaenoic acid, lactoferrin, nucleotides and other nutrients.

Effects of food enriched with egg yolk hydrolysate (bone peptide) on bone metabolism in orchidectomized dogs.

We examined the effects of chicken egg hydrolysate (also known as "bone peptide" or BP) on bone metabolism in 5- to 8-month-old orchidectomized dogs. The bone formation marker serum bone alkaline phosphatase (BAP) and the bone resorption marker urine deoxypyridinoline (DPD) were used as indicators to measure changes in bone metabolism. The following results were observed that Serum BAP was higher in dogs fed BP-enriched food throughout the clinical investigation. Serum BAP was statistically significantly higher in dogs fed BP-enriched food than in dogs fed non-BP-enriched food at 2 months after orchidectomy. This suggests that BP promoted bone formation immediately after orchidectomy.

Comparative investigation of body composition in male dogs using CT and body fat analysis software.

In small animal veterinary practices, body condition score (BCS) is generally used to diagnose obesity. However, BCS does not constitute objective data. In this study, we investigated the value of using human body fat analysis software for male dogs. We also compared changes in body fat after neutering. Changes in body fat at the time of neutering (age 1 year) and 1 year later were compared by performing CT scanning and using human body fat analysis software. We found that body fat increased in all the individuals tested. In terms of the site of fat accumulation, subcutaneous fat was more pronounced than visceral fat with a marked increase on the dorsal side of the abdomen rather than the thorax.

Standard electrocardiographic data of young Japanese monkeys (Macaca fusucata).

The electrocardiogram of nonhuman primates is similar to that of humans because of similar intrathoracic heart position and structure. Despite the frequent use of nonhuman primates in biologic studies, few electrocardiographic studies of Japanese monkeys (Macaca fusucata) have been reported, and no reference data are available for this species. We obtained limb-lead electrocardiograms from indoor-bred and housed ketamine-sedated Japanese macaques (48 male; 56 female; mean age, 44.3 mo; mean body weight, 4.84 kg) in the dorsal recumbency. The following quantitative data was obtained: heart rate, P wave amplitude and width, R wave amplitude, QRS duration, PR interval, QT interval, T wave height, and mean electrical axis. Corrected QT intervals were calculated by using the Bazett and Fridericia formulae. Measurements were evaluated according to sex and age. The duration of the QRS complex showed moderate correlation with age in male monkeys. All parameters, except heart rate, were similar to previous reports from Japanese, cynomolgus, and other macaques. P waves, R waves and mean electrical axis did not differ significantly between humans and Japanese macaques, but the wave amplitude in macaques was half that in humans. Our electrocardiographic measurements can serve as normal reference data for sedated, young Japanese monkeys.

Identification of ventrolateral intramedullary intervertebral disc herniation in a dog.

A 10-year-old male cross-breed dog was brought to Nihon University Animal Hospital with a history of acute onset of paralysis in the pelvic limbs 13 days previously. Magnetic resonance imaging revealed an intramedullary linear tract in the spinal cord at the thoracic vertebrae 12-13 region, which appeared hyperintense on T2-weighted images, but was hypointense and isointense on T1-weighted images when compared with normal parenchyma of the spinal cord. A hemilaminectomy was performed and a blob of what appeared to be fibrous tissue was found adhering to the surface of the dura mater. The diameter of the blob was about 4 mm. A durotomy was performed over the affected area and chondroid material was found within the spinal cord. Material from the nucleus pulposus penetrated the dura mater from the ventral aspect of the spinal cord in previously reported intramedullary intervertebral disc herniation cases, but, in this case, penetration occurred from the left ventrolateral aspect and progressed through to the right lateral aspect, forming a visible blob of what appeared to be fibrous tissue on the surface of the dura mater at the exit point. To the best of our knowledge, this was the first case report of an intramedullary intervertebral disc herniation originating from the ventrolateral aspect of the spinal cord in a dog.

Gastric hypomotility in chronic upper gastrointestinal disease of Japanese macaques (Macaca fuscata).

Japanese macaques bred indoor for laboratory use often show chronic anorexia and intermittent vomiting. In some of our macaques gastric air was observed on physical examination, and we suspected abnormality of gastric motility. We therefore performed contrast radiographic examinations of the gastrointestinal tract without anesthesia of 8 macaques with gastrointestinal symptoms and 9 asymptomatic controls from the same laboratory. Changes of abdominal radiography over time were observed following oral administration of contrast medium. In all control animals, contrast medium had completely passed from the stomach within 150 min after administration. However, all animals with gastrointestinal symptoms retained some contrast medium in the stomach. Gastric emptying time of contrast medium was associated with excessive gastric air in Japanese macaques; therefore, gastric emptying time seems to be associated with decreased gastric motility.

Magnetic resonance imaging features and clinical signs associated with presumptive and confirmed progressive myelomalacia in dogs: 12 cases (1997-2008).

OBJECTIVE: To characterize clinical signs and findings from magnetic resonance imaging (MRI) and CSF analysis for dogs with progressive myelomalacia (PM) diagnosed on the basis of clinical and histologic features. DESIGN: Retrospective case series. ANIMALS: 5 dogs with confirmed PM and 7 with presumptive PM evaluated from 1997 through 2008. PROCEDURES: Medical records of study dogs were reviewed, and clinical signs and MRI and CSF findings were evaluated. A clinical diagnosis of PM was made on the basis of detection of disk-associated spinal cord compression via MRI and progression of clinical signs from initial paraparesis or paraplegia to thoracic limb lower motor neuron paresis to tetraplegia associated with cranial migration of the extent of cutaneous trunci reflex extinction and analgesia, terminating in death due to respiratory paralysis. RESULTS: All dogs were paraplegic and had signs of lower motor neuron lesions. As revealed by MRI of the vertebral column, the length of abnormal signal intensity was more than 6-fold, compared with the body length of L2. In some dogs, these abnormal MRI findings were observed before PM was clinically diagnosed. The CSF examination revealed hemorrhagic xanthochromia. CONCLUSIONS AND CLINICAL RELEVANCE: A hyperintense region of the spinal cord > 6 times the length of L2 on T2-weighted imaging might be a characteristic MRI finding of PM. In some dogs, such MRI findings were observed before PM was clinically diagnosed. Progressive myelomalacia may thus be diagnosable at the early stage through MRI and CSF examination before signs of PM develop.

Establishment of vertebral heart scale in the growth period of the Japanese macaque (Macaca fuscata).

We performed a thoracic X-ray examination of 56 Japanese macaques to obtain normal reference values for vertebral heart scale (VHS). Mean VHS was 10.25 +/- 0.94 v. In males, mean VHS was 10.56 +/- 0.73 v, with no significant correlation to age or weight. In contrast, mean VHS in females was 9.97 +/- 1.03 v, and tended to decrease with increasing age and weight. These findings will facilitate the diagnosis of cardiac disease in Japanese macaques in the future.

MRI of secondary cervical syringomyelia in four cats.

This report describes the use of magnetic resonance imaging (MRI) to diagnose cervical syringomyelia in 4 cats. MRI revealed enlargement of the lateral ventricle in all the cats. Of the 4 cases, MRI revealed herniation of the cerebellum in 3 cats, an isolated fourth ventricle in 1 cat, severe hydrocephalus in 2 cats and brain masses in 1 cat. In this report, the cervical syringomyelia in these cats may have been due to formation of a secondary syrinx (enlargement of the central canal) as a result of blockage of flow in the outlet of the fourth ventricle caused by FIP encephalomyelitis or secondary cerebellar tonsillar herniation caused by increased intracranial pressure due to intracranial masses or may have been due to caudal compression of the cerebellum caused by increased intracranial pressure due to hydrocephalus.

Magnetic resonance imaging and computed tomography findings of vertebral osteosarcoma in a cat.

An 8-year-old female Persian cat with a gait disorder was brought to our hospital. Pelvic limb mobility had gradually reduced over the preceding 3 months, then rapidly deteriorated 2 weeks before consultation. Signs also occurred in the thoracic limbs. With a tentative diagnosis of neural disease, magnetic resonance imaging and computed tomography were performed. T1-weighted imaging showed isointensity in the seventh cervical vertebra, while T2-weighted imaging revealed hypointensity. Contrast-enhanced T1-weighted imaging revealed a uniformly enhancing mass. Extirpation of the mass relieved the clinical signs, leading to disappearance of the neurological signs. The histopathological examination suggested osteosarcoma.

T cell receptor repertoire in BALB/c mice varies according to tissue type, sex, age, and hydrocortisone treatment.

Diversity in T cell recognition of antigens is determined by diverse usage of T cell receptor (TCR) repertoire. TCR repertoire analysis provides fundamental information for understanding T cell immune responses in the pathogenesis of various diseases. In the present study, we examined the TCR repertoire in various tissues in normal BALB/c mice. The TCR alpha chain variable region repertoires were consistent among the spleen, lymph nodes, and the thymus. The TCR beta chain variable region (TCRBV) repertoires were consistent between the spleen and lymph nodes, but different in the thymus. The TCR repertoires also differed in the lungs and the intestinal tract. The TCR repertoires were consistent between male and female mice, except for TCRBV15-1. TCR repertoire was almost similar in 3- and 7-week-old mice, except for TCRBV1-1, 8-3, and 14-1. The present findings suggest that the TCR repertoire of mice varies according to tissue type, sex and age. Additional analysis of the TCR repertoire, i.e., the effect of hydrocortisone (HC), was carried out. After the HC treatment, although the thymic T cells decreased to one-tenth, only a small fraction of CD4(+)CD8(+) T cells survived the treatment. Furthermore, the percentages of thymic T cells bearing TCRBV3-1, 5-1, 5-2, and 16-1 substantially decreased, but the percentage of cells bearing TCRBV12-1 did not decrease. The present findings suggest that the HC susceptibility of immature thymic T cells is different between TCR families.

Ocular granulomatous meningoencephalomyelitis in a dog: magnetic resonance images and clinical findings.

A 4-year-old female miniature dachshund presented with a history of progressive decrease in vision, neck pain, and ataxia for which an MRI was performed 21 days after the initial consultation. The optic nerves showed isointensities on T1- (T1W) and T2-weighted (T2W) images that were enhanced by the contrast medium. The optic chiasm was swollen. Other parts of the cerebral parenchyma, not only the visual pathway, showed symmetrical hyperintensities on T2W images. Cerebrospinal fluid showed increased anti-GFAP autoantibodies. The dog received corticosteroid therapy despite which she exhibited anorexia, vomiting, and lethargy. Hence, a follow-up MRI was repeated 30 days after the initial consultation in which T2W images showed enlargement of the hyperintense area. The dog died 45 days after the initial consultation. Postmortem pathological examination confirmed a diagnosis of granulomatous meningoencephalomyelitis (GME).

Regeneration of intervertebral disc by the intradiscal application of cross-linked hyaluronate hydrogel and cross-linked chondroitin sulfate hydrogel in a rabbit model of intervertebral disc injury.

The degeneration of intervertebral disc (IVD) is a major cause of low back pain. However, there is no satisfactory preventive treatment for degenerative disc disease (DDD). In this study, we examined the effects of a novel cross-linked hyaluronate hydrogel and cross-linked chondroitin sulfate (CS) hydrogel on a rabbit model of IVD injury. We injected 300 microl of phosphate buffer saline, 1% sodium hyaluronate, cross-linked hyaluronate hydrogel, or cross-linked CS hydrogel into the injured IVDs. One, three or six months after treatment, the whole spinal columns were dissected and magnetic resonance (MR) images of the IVDs were examined. It was noted that the IVD, which was injected with cross-linked hyaluronate hydrogel or cross-linked CS hydrogel mostly retained the normal signal intensity of the MR images. These IVDs exhibited a higher degree of staining with safranin-O than the control discs or 1% sodium hyaluronate-injected discs, suggesting that the intradiscal application of cross-linked hyaluronate hydrogel or cross-linked CS hydrogel probably inhibits the degenerative cascade of the DDD. The intradiscal administration of these drugs is safe, easy and costs less. In the near future, these intradiscal injections may become the standard therapy for the treatment of DDD instead of the spine surgeries.